| Chiral compounds play an important role in the fields of medicine, fine chemical, life science, material science and so on. A considerable amount of used drugs are chiral, chiral aromatic alcohols are great importance intermediates for the synthesis of chiral drugs, so it is very useful in the field of pharmacy. Bio-catalytic reactions under mild conditions, good catalytic efficiency, high stereoselectivity, less pollution and environmentally friendly, it can complete some reactions which chemical method is difficult or impossible to complete.In this research, asymmetric reduction of acetophenone and its derivant to prepare chiral aromatic alcohols by microbiological method. The analytical methods of qualitative and quantitative for determining chiral alcohols in the system of biodegradation were established by HPLC and GC. The main research contents and results are shown as follows:1. A simultaneous quantitative analytical method of2-methylacetophenone and2-methyl-1-phenylethanol was set by GC. The detector was FID, the column was chiral capillary column, the injection temperature was220℃and the detector temperature was220℃. Using temperature-programmed, the initial temperature was100℃,5℃·min-1rose to155℃, then2℃·min-1rose to160℃.The result showed that the linear correlation ccoefficient of (R)-2-methyl-l-phenylethanol was0.9994in0.045～4.45mg-mL-1, and that (S)-2-methyl-l-phenylethanol was0.9995in0.044～4.36mg-mL-1,2-methylacetophenone was0.9995in0.53～10.52mg-mL-1. The relative standard deviation was less than1.7%and the average recoveries were from100.6%to107.2%. This method was simple, accurate and reliable.A simultaneous quantitative analytical method of4-methylacetophenone and4-methyl-1-phenylethyl alcohol was set by HPLC. The best chromatographic conditions were chiralce OJ-RH, acetonitrile-water (30:70) as the mobile phase, the UV detection wavelength was220nm and at a low rate of0.5mL·min-1.A simultaneous quantitative analytical method of4-chloroacetophenone and4-chloro-l- phenylethyl alcohol was set by GC. The detector was FID, the column was chiral capillary column, the injection temperature was220℃, the detector temperature was220℃. Using temperature-programmed, the initial temperature was120℃,5℃·min-1rose to165℃, then maintained2min,5℃·min-1rose to200℃2. Thirteen strains for asymmetric reduction of4-methylacetophenone were screened from soil samples using4-methylacetophenone as the sole carbon source. The strain T19with good catalytic efficiency which can asymmetric reduced of4-methylacetophenone to S-4-methyl-phenylethyl alcohol. T19was identified as Mucor circinelloides according to the16S rDNA sequences, it was designated as Mucor circinelloides T19.3. The characteristics of (S)-4-methyl-phenylethyl alcohol production from4-methylacetophenone by Mucor circinelloides T19were studied. The results showed that when pH=6-7,30℃and50g-L-1glucose was added as the cosubstrate, the addition of4-methylacetophenone was3g·L-1. The conversion of4-methylacetophenone was reached98.9%and e.e. was100%.4. The research showed that Mucor circinelloides T19can also asymmetric reduce of acetophenone, propiophenone,2-methylacetophenone,4-chloroacetophenone and4-fluoroacetophenone to corresponding S-aromatic alcohols with high conversion and stereoselectivity. Discoveried that Mucor circinelloides T19had different catalytic efficiency and stereoselectivity to different structure of aromatic ketone.5. Add acetophenone, at the same time, the4-methyl-acetophenone,2-methyl acetophenone,4-chloroacetophenone,4-fluorophenyl were added respectively to compose binary substrate system in order to study the competitive reaction of acetophenone and these aromatic ketones in the catalysis of T19.(1) In the concomitant binary substrate system of acetophenone and4-methylacetophenone,4-methylacetophenone had obvious reaction advantage, the conversion of4-methylacetophenone (61.1%-84.4%) were greater than the conversiong of acetophenone (39.5%～81.5%). The ee values of (S)-4-methyl-phenylethyl alcohol (100%) reduction product greater than the ee values of (S)-1-phenylethyl alcohol (about86%).(2) In the concomitant binary substrate system of acetophenone and2-methylacetophenone, the conversion of acetophenone (77.2%) were greater than the conversiong of2-methylacetophenone (55.0%). The stereoselective of acetophenone (ee value of (S)-1-phenylethyl alcohol was about82%) and2-methylacetophenone (ee value of (S)-2-methyl-l-phenylethanol was about88%).(3) In the concomitant binary substrate system of acetophenone and4-chloroacetophenone,4-chloroacetophenone had obvious reaction advantage. The reaction rate of4-chloroacetophenone was greater than acetophenone, the ee values of (S)-chloro-1- phenylethyl alcohol (about97%) reduction product greater than the ee values of (S)-l-phenylethyl alcohol (about74%).(4) In the concomitant binary substrate system of acetophenone and4-fluoroacetophenone, their conversion rates were roughly equal to each other which were catalyticed by T19. But the ee values of (S)-fluoro-1-phenylethyl alcohol (about94%) was greater than (S)-1-phenylethyl (about87%). |
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